Expressão Recombinante de uma Miraculina de Citrus em Pichia pastoris e em Escherichia coli

Abstract

The high consumption of sugar in Brazil and in the world result in drastic consequences for human health, causing numerous diseases. In the search for alternatives to reduce sugar consumption, several studies have emerged related to the development of caloric and non-caloric sweeteners, of natural or artificial origin. Among the natural compounds there is a group of proteins that have the property of modifying the flavor of food. Within this class of molecules, there is the protein called miraculin, from the plant Richardella dulcifica, native to Africa, which has the property of modifying the acidic taste of food into sweet. Due to this potential, there are reports of several tests carried out in an attempt to produce miraculin through recombinant expression in other organisms. However, other studies have discovered the existence of miraculin-like proteins in other plants, although there is still no description of miraculin-like proteins with such property. On the other hand, in silico analyzes allowed finding in Citrus paradisi a protein with a gene sequence with high similarity to that found in the original miraculin. Therefore, the characterization of this protein and the exploration of its potential ability to modify flavor may result in non-caloric alternatives to sweeten acidic foods, thus contributing to an improvement in human health. Therefore, the objective was the recombinant expression of a miraculin derived from Citrus paradisi, called Citros_Mir, in Pichia pastoris yeast and in Escherichia coli bacteria. Cloning was performed in the propagation vector pGEM-T. Expression in E. coli occur with pET-28a expression vector and the Rosetta strain, in addition to testing temperatures of 20, 30 and 37ºC, and final concentrations of 0.2; 0.4 and 1.0 mM of the IPTG expression inducer. Expression in P. pastoris was performed with the pPICZαC expression vector and the X-33 and KM71H strains. In this system, was test the final concentrations 0.75, 1, 2 and 3% of the methanol expression inducer, concluding that the yeast presents a better expression of proteins in a culture medium containing 2% of methanol. The results obtained showed that attempts to express Citros_Mir in these systems were not efficient, suggesting the need for new tests, modifying conditions and/or expression systems.